Summary/Abstract Human granulocytic anaplasmosis is the second most common tick-borne disease in the United States. The causative agent, Anaplasma phagocytophilum, is an obligate intracellular pathogen that resides in 2 different locations during its life cycle -- mammalian neutrophils and Ixodes scapularis salivary glands. This project investigates whether tick genes that are up- or down-regulated in the presence of A. phagocytophilum play important roles in infection. These studies build upon our efforts showing that A. phagocytophilum alters host gene expression and uses specific molecules for survival. As examples, in mammals Anaplasma requires fucosylation of host proteins for infection, and represses gp91phox in neutrophils to prevent the respiratory burst and thereby aid survival. In ticks Anaplasma induces I. scapularis salivary protein (Salp) 16 to help colonize the vector by mechanisms that are currently not known. In addition, our preliminary data show that A. phagocytophilum infection of ticks decreases the expression of I. scapularis salp10 and salp17, two genes with unknown functions. We now hypothesize that fucosylation is important for A. phagocytophilum infection of I. scapularis and that Salp16, an Anaplasma-induced tick gene, may be a substrate of fucosylation. This is based, in part, on our preliminary data showing that RNA interference-mediated knockdown of a putative tick a-1,3 fucosyltransferase decreases A. phagocytophilum infection of I. scapularis, and that Salp16 has the potential to be fucosylated. We also hypothesize that salp10 and salp17, two tick genes that are repressed during A. phagocytophilum infection of I. scapularis are also important in the Anaplasma life cycle. We will therefore characterize salp10 and salp17, and use a lentiviral-based system to introduce these genes into ticks -- the first [unreadable]knock-in[unreadable] studies to generate ticks that constitutively express a specific gene. We postulate that A. phagocytophilum survival will be altered in ticks when salp10 and salp17 are not normally down-regulated during infection. These studies will provide an understanding of how I. scapularis genes that are up- or down-regulated in the presence of A. phagocytophilum are important in infection, delineate the similarities and differences between infection in the arthropod vector and mammalian host, and lead to new strategies to interfere with the Anaplasma life cycle.